1-O-Acetyl-β-D-galactopyranose: A novel substrate for the transglycosylation reaction catalyzed by the β-galactosidase from Penicillium sp.

Article abstract of DOI:10.1016/S0008-6215(02)00027-7

1-O-Acetyl-β-D-galactopyranose (AcGal), a new substrate for β-galactosidase, was synthesized in a stereoselective manner by the trichloroacetimidate procedure. Kinetic parameters (K_M and k_cat) for the hydrolysis of 1-O-acetyl-β-D-galactopyranose catalyzed by the β-D-galactosidase from Penicillium sp. were compared with similar characteristics for a number of natural and synthetic substrates. The value for k_cat in the hydrolysis of AcGal was three orders of magnitude greater than for other known substrates. The β-galactosidase hydrolyzes AcGal with retention of anomeric configuration. The transglycosylation activity of the β-D-galactosidase in the reaction of AcGal and methyl β-D-galactopyranoside (1) as substrates was investigated by ¹H NMR spectroscopy and HPLC techniques. The transglycosylation product using AcGal as a substrate was β-D-galactopyranosyl-(1→6)-1-O-acetyl-β-D-galactopyranose (with a yield of ～70%). In the case of 1 as a substrate, the main transglycosylation product was methyl β-D-galactopyranosyl-(1→6)-β-D-galactopyranoside. Methyl β-D-galactopyranosyl-(1→3)-β-D-galactopyranoside was found to be minor product in the latter reaction.

Full text of DOI:10.1016/S0008-6215(02)00027-7

Carbohydrate Research 337 (2002) 635–642
www.elsevier.com/locate/carres
Note
1-O-Acetyl-b- -galactopyranose: a novel substrate for the
D
transglycosylation reaction catalyzed by the b-galactosidase from
Penicillium sp.
Alexander I. Zinin,a Elena V. Eneyskaya,b Konstantin A. Shabalin,b Anna
A. Kulminskaya,b Sergei M. Shishlyannikov,b Kirill N. Neustroevb,*
aN.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky a6. 47, Moscow 119991, Russia
bPetersburg Nuclear Physics Institute, Russian Academy of Science, Molecular and Radiation Biology Di6ision, Gatchina, St. Petersburg 188350,
Russia
Received 9 August 2001; accepted 10 January 2002
Abstract
1-O-Acetyl-b-
the trichloroacetimidate procedure. Kinetic parameters (KM and kcat) for the hydrolysis of 1-O-acetyl-b-
catalyzed by the b- -galactosidase from Penicillium sp. were compared with similar characteristics for a number of natural and
D
-galactopyranose (AcGal), a new substrate for b-galactosidase, was synthesized in a stereoselective manner by
D
-galactopyranose
D
synthetic substrates. The value for kcat in the hydrolysis of AcGal was three orders of magnitude greater than for other known
substrates. The b-galactosidase hydrolyzes AcGal with retention of anomeric configuration. The transglycosylation activity of the
b-
spectroscopy and HPLC techniques. The transglycosylation product using AcGal as a substrate was b-
1-O-acetyl-b- -galactopyranose (with a yield of ꢀ70%). In the case of 1 as a substrate, the main transglycosylation product was
methyl b- -galactopyranosyl-(1“6)-b- -galactopyranoside. Methyl b- -galactopyranosyl-(1“3)-b- -galactopyranoside was
found to be minor product in the latter reaction. © 2002 Elsevier Science Ltd. All rights reserved.
D
-galactosidase in the reaction of AcGal and methyl b-D
-galactopyranoside (1) as substrates was investigated by 1H NMR
D
-galactopyranosyl-(1“6)-
D
D
D
D
D
Keywords: b-
D
-Galactosidase; Penicillium sp.; 1-O-Acetyl-b-D-galactopyranose; Transglycosylation; Disaccharides
dases are frequently used in the preparation of a variety
of transglycosylation products.7,8
1. Introduction
b-D-Galactosidases from various sources have been
In recent years, enzymatic syntheses using glycosi-
dases that possess transglycosylation activity are be-
coming more common.1,2 This approach has the
advantage of regioselective and stereoselective reactions
without the protection and deprotection processes re-
quired with synthetic chemical methods.3,4 Enzymatic
synthesis has been utilized to obtain neoglycoproteins
and neoglycoconjugates for use in medicine and other
fields.5,6 Enzymatic transglycosylation, the reverse of
hydrolysis effected by hydrolases, has been more widely
used than glycosyltransferase reactions since glycosi-
successfully applied in the enzymatic syntheses via
transglycosylation of biologically active galac-
tooligosaccharides. The b-
circulans was used in the synthesis of N-acetyllac-
tosamine [b- -Gal-(1“4)- -GlcNAc], the main com-
ponent of oligosaccharides from human milk.9
Transglycosylation activity of the b- -galactosidases
from several sources was also used in the synthesis of
D-galactosidase from Bacillus
D
D
D
b-
D
-Gal-(1“3)-D
-GlcNAc,10,11 the key oligosaccharide
of sialyl Lewis a (sLea), which has been reported to be
the ligand involved in the metastasis of cancer cells.12
Also of note, b-
nent of the mucin-type glycoprotein side chains.13 Fi-
-galactosidase from E. coli was employed
D
-Gal-(1“3)-
D-GlcNAc is a compo-
* Corresponding author. Tel.: +7-812-7132014; fax: +7-
812-7132303.
E-mail address: neustk@omrb.pnpi.spb.ru (K.N. Neu-
stroev).
nally, the b-
D
in the enzymatic synthesis of some di-b-D-galactosyl
glycopeptides.14
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00027-7

636
A.I. Zinin et al. / Carbohydrate Research 337 (2002) 635–642
The choice of donor in the transglycosylation reac-
under neutral conditions (such as Bn, chloroacetyl) is
needed because of the hydrolytic liability of an
anomeric acyl group. Second, if a specific anomer is
required, the acyl group should be introduced stereose-
lectively or, at least, the two anomers should be readily
separable.
tions is important, as it affects the yield of the product.
Therefore, the search for new donors, as well as new
glycosidases having high transglycosylation activity, is
essential for increasing the yield and improving the
stereospecificity of the reaction.15,16
In this work, we describe the synthesis of 1-O-acetyl-
Per(chloroacetylated) glucopyranosyl bromide reacts
with silver carboxylates in CH2Cl2 to give 1-O-acyl
derivatives in good yield and with excellent b-selectiv-
ity, although the yields at the deprotection step are
moderate.18 Most of the known 1-O-acyl derivatives of
glucose were obtained via acylation of 2,3,4,6-tetra-O-
b-
-galactosidases, and the transglycosylation reactions
of this substrate. To evaluate the suitability of this
compound for enzymatic syntheses, we used the b-
galactosidase (b- -galactoside galactohydrolase, EC
D
-galactopyranose (AcGal), a novel substrate for b-
D
D
-
D
3.2.1.23) having transglycosylation activity, isolated
from filamentous fungi Penicillium sp. and for which
preliminary investigation by X-ray crystallography has
been reported.17
benzyl-
tivity. The reaction of ethyl 2,3,4,6-tetra-O-benzyl-
1-thio-b- -galactopyranoside with N-iodosuccinimide
D-glucopyranose, usually with poor stereoselec-
D
and AcOH in CH2Cl2 was reported to give the 1-acetate
in excellent yield as a 1:1 mixture of anomers that was
not separated.19
The reaction of O-glycosyl trichloroacetimidates with
carboxylic acids in a non-polar solvent affords 1-O-acyl
sugars in high yield and complete inversion of configu-
ration.20,21 Since both anomers of 2,3,4,6-tetra-O-ben-
2. Results and discussion
Synthesis of 1-O-acetyl-i-D-galactopyranose.—To
obtain a 1-O-acylated sugar, two distinct problems
must be solved. First, a protective group removable
zyl-
readily available,22,23 we chose to apply the trichloro-
acetimidate method for synthesis of 1-O-acetyl-b-
galactopyranose. Action of AcOH on 2,3,4,6-tetra-O-
-galactopyranosyl 2,2,2-trichloroacetimid-
D-galactopyranosyl 2,2,2-trichloroacetimidate are
D
-
benzyl-a-
D
ate22 in CH2Cl2 afforded the 1-O-acetylated product in
76% yield following column chromatography. To our
surprise it contained significant amounts of the a
anomer (10:1 b/a by NMR), non-detectable by TLC
because of small differences in Rf, irrespective of the
eluent used. We were able to isolate the desired b
anomer by crystallization, which lowered the yield to
51%. Hydrogenolytic debenzylation gave crystalline 1-
O-acetyl-b-D-galactopyranose in quantitative yield.
Analysis of the enzymatic hydrolysis of 1-O-acetyl-i-
-galactopyranose.—The b-galactosidase from Penicil-
D
lium sp. catalyzes the hydrolysis of 1-O-acetyl-b-
D-gal-
actopyranose. -Galactose and acetic acid were found
D
Fig. 1. Analysis of the hydrolysis of AcGal as a function of its
concentration by the Lineweaver–Burk method: ꢀ, data
obtained by the HPLC method; ꢁ, NMR data.
1
by direct H NMR examination to be the products of
the hydrolysis (not accounting for the transglycosyla-
tion process). As shown in Fig. 1, the dependence of the
initial hydrolytic rate on the substrate concentration
over the range from 10 to 1 mM obeys Michaelis–
Menten kinetics and can be expressed by Lineweaver–
Burk presentation of the data. The kinetic parameters
Km and kcat (Table 1) were evaluated by direct analysis
Table 1
Kinetic parameters of reactions catalyzed by b-galactosidase
from Penicllium sp.
Substrate
Km (mM) kcat (mmol
min−1 mg−1
)
1
of the reaction course in H NMR experiments and by
HPLC analysis of liberated D-galactose, as described in
p-Nitrophenyl
1.0
0.125
the Section 3. Independent experiments showed that
spontaneous breakage of the O-acetyl linkage in AcGal
did not take place under the conditions of enzymatic
hydrolysis. AcGal was found to be a competitive in-
hibitor in the two-substrate process of hydrolysis of
b-
D
-galactopyranoside
-galactopyranoside (1) 5.9
Methyl b-
D
0.00067
14
1-O-Acetyl-b-
(AcGal)
D
-galactopyranose
3.9
Lactose
11.1
0.04
p-nitrophenyl b- -galactopyranoside (2), with the inhi-
D

A.I. Zinin et al. / Carbohydrate Research 337 (2002) 635–642
637
b anomer of D-galactose is formed at the initial stage of
the hydrolysis. At later stages, the a anomer appears as
a result of mutarotation. Mutarotation was considered
to be complete when the anomeric ratio (:30% a,
:70%b) was established (ꢀ20 h).25 These data un-
equivocally demonstrate that hydrolysis catalyzed by
the enzyme proceeds with the retention of the anomeric
configuration.
Analysis of steady-state kinetics of the hydrolytic re-
action catalyzed by the i-D-galactosidase.—Kinetics of
the b- -galactosidase hydrolysis of 1, lactose, and 2
D
obeys Lineweaver–Burk equations in the concentration
range from 20 to 5 mM for lactose, from 20 to 2 mM
for 1, and from 2 to 0.2 mM for 2 (Table 1). As may be
seen from the data, the kcat value in the hydrolysis of
AcGal is 350 times higher than the corresponding value
for lactose and 17,500 times higher than for 1. It is
about 100 times higher than the maximal velocity for
the ‘fastest’ b-galacosidase substrate, 2. At the same
time, values for KM in the hydrolysis of AcGal and
galactooligosaccharides are quite close.
Fig. 2. pH-dependence of the b-
D-galactosidase activity to-
ward AcGal (ꢂ), 1 (ꢀ), 2 (ꢃ).
Glycosidases operate by
a double-displacement
mechanism in which a glycosyl–enzyme intermediate is
formed first during the glycosylation step, followed by
hydrolysis (deglycosylation step) in a general acid–
base-catalyzed process through oxocarbenium ion-like
transition states.26 As just shown, the Penicillium b-
D-
galactosidase acts via a double-displacement mecha-
nism.27 A minimal kinetic scheme describing the
two-step process of the hydrolysis (not taking into
account transglycosylation reactions) can be written:
Fig. 3. Time course of
formation during the hydrolysis of AcGal.
D
-galactose a (ꢂ) and b anomer (ꢁ)
bition constant (4.1 mM) close to the Km value for the
hydrolysis of AcGal (Table 1). Consequently, we can
conclude that AcGal binds to the normal catalytic
center b-D-galactosidase. As shown in Fig. 2, the pH
optimum for the hydrolysis of AcGal coincides with
where E is the enzyme; S is the substrate; ES is the
enzyme–substrate complex (Michaelis complex); ES% is
the intermediate galactosyl–enzyme complex; P1 is the
reaction product formed from the aglycon portion of
the substrate; P2 is
D-galactose; k1, k−1, k2, and k5 are
that of the well-known substrates, 1 and 2.
Stereochemical course of action.—Application of H
1
the rate constants of the corresponding stages (k5=
k%·[H2O]). The solution to this scheme under the condi-
NMR provides a direct approach for determining the
stereochemical course of hydrolysis catalyzed by gly-
canases.24 Chemical shifts and coupling constants of the
anomeric protons in a- and b-galactosides and in the
product hemiacetals are distinct and readily observed.
When sufficient quantities of the b-galactosidase was
used to complete the hydrolysis of AcGal within 15
min, the initially formed b anomer could be detected
before mutarotation had occurred to any significant
extent. The time-course of the appearance of the a and
5
tions of steady-state ES and ES% complexes can be
readily determined from the Michaelis–Menten
equation:
d[P1] d[P2] kcat[E0][S]
w=
=
=
(1)
dt
dt
Km+[S]
where w is the initial reaction rate:
k2k5
k2+k5
kcat
=
(2)
b anomers of
D-galactose resulting from hydrolysis of
AcGal with the b-galactosidase from Penicillium is pre-
On the assumption of k2ꢁk5, the value for kcat=k2 for
‘fast’ substrates. This particular scheme was successfully
sented in Fig. 3. From these data, it is apparent that the

638
A.I. Zinin et al. / Carbohydrate Research 337 (2002) 635–642
applied to describe the kinetic properties of an a-D-
galactosidase from Phanerochaete chrysosporium,28
Bacillus (1“3)-(1“4)-b-glucanase,29 and the a-galac-
tosidase from Trichoderma reesei.30 It was established
that there are two types of substrates for a-
D-galactosi-
dase: ‘fast’ (p-nitrophenyl a- -galactopyranoside) and
D
‘slow’ (melibiose, raffinose, etc.).30 Differentiation into
fast and slow substrates was based on the dissociation
constants for the enzyme–substrate complex at the
stage of releasing the reaction product, which is the
second stage of the two-step hydrolysis process.
Here, AcGal might be considered as a ‘fast’ substrate
along with 2, in contrast to ‘slow’ lactose and 1. The
stage of deglycosylation of the ES% complex is the same
for both ‘slow’ and ‘fast’ substrates, and only the
difference in k2 values, the formation-rate constant for
the ES% complex, affects the kcat values for different
substrates. Thus, in terms of the kinetic scheme pre-
sented, our data indicate that acetic acid is the best
leaving group when compared with MeOH and p-nitro-
phenol. Using Eq. (4) from Section 3, we found that the
k+H value for acid hydrolysis of 1 is about 35 times
lower than for AcGal (1.76 and 0.05 M−1 min−1, for
AcGal and 1, respectively). Due to the acid–base na-
ture of the b-galactosidase catalysis in this case, one
possible explanation for the relatively high k2 value for
AcGal is the low stability of the substrate under acid
conditions.31
Fig. 4. Formation of transglycosylation products in the hy-
drolysis of AcGal at 6.9 (ꢁ), 13.8 (ꢂ), and 27.5 (ꢃ) mM
concentration of the substrate as a function of time.
Transglycosylating acti6ity of the i-D-galactosidase
with AcGal and 1 as substrates.—Transglycosylating
activity in the reaction of substrate transglycosylation
was investigated with AcGal as a substrate. Kinetics of
formation of the transglycosylation product measured
1
by H NMR at different concentrations of AcGal is
Fig. 5. Time-dependence of AcGal hydrolysis (AcGal concen-
tration of 60 mM): ꢂ, AcGal; ꢁ, acetic acid; ꢄ, transglyco-
shown in Fig. 4. The time-course of the hydrolysis of
AcGal at a concentration of 60 mM assayed by the
same method is presented in Fig. 5. Based on these
data, we used Eq. (3)32 to estimate the autocondensa-
tion reaction yield.
sylation product; ꢃ,
D-galactose.
Transfer product
Transfer product+D-galactose
Transfer yield=
(3)
As seen from Fig. 6, the yield of transglycosylation
product in the condensation reaction with AcGal is
ꢀ70% during the first 10 min of reaction. The corre-
sponding value for the same concentration of 1, also
1
obtained using the H NMR technique, was roughly
45%. This would suggest that AcGal may be a better
donor for enzymatic synthesis that involves a transgly-
cosylation reaction. It should be noted that the yield of
70% is rather high in comparison with exo-glycosidases
from other sources exhibiting transglycosylating activ-
ity. The yield in the substrate transglycosylation reac-
tions catalyzed by a-galactosidases from thermophilic
sources was 20%;33 this value for E. coli b-
D
-galactosi-
Fig. 6. Time-dependence of transglycosylation product yield
in the hydrolysis of AcGal at 60 mM concentration.
dase was 20–30%,34 and for a-galactosidase from

A.I. Zinin et al. / Carbohydrate Research 337 (2002) 635–642
3. Experimental
639
Chemicals.—Methyl b-
D
-galactopyranoside, p-nitro-
phenyl b-
D
-galactopyranoside, lactose, b-
D
-galactopy-
ranosyl-(1“6)-D-galactopyranose, salts, and bovine
serum albumin (BSA) were purchased from Sigma, St.
Louis, MO, reagents used for synthesis of AcGal and
solvents were from Fluka and E. Merck (Germany).
General methods.—Protein concentrations were mea-
sured following the Lowry procedure with BSA as the
standard.37 Optical rotations were determined with a
1
Jasco DIP-360 polarimeter. All H and 13C NMR spec-
tra were recorded with an AMX-500 Bruker spectrome-
ter. Prior to NMR analysis, the b-galactosidase, buffer
materials, and substrates were freeze-dried twice from
D2O. The 1H NMR measurements were made with
MeCN as the internal standard, l 2.1 ppm. To measure
13C chemical shifts, the methyl acetone resonance at l
30.2 ppm was used as an internal standard. Oligosac-
charides, products of enzymatic hydrolysis, and prod-
ucts of transglycosylation were analyzed qualitatively
by thin-layer chromatography on Kieselgel 60 plates (E.
Merck) with a mobile phase of 2:2:1 ethanol–butan-1-
ol–water. Plates were developed at room temperature,
air dried, and sprayed with 5% H2SO4 in 2-propanol,
followed by incubation at 120 °C for 8 min.
Fig. 7. pH-dependence of formation of transglycosylation
products in the condensation reaction with AcGal (ꢀ) and 1
(ꢁ).
Bifidobactericum adolescentic −15%.35 As may be seen
from the kinetics of product formation for the transgly-
cosylation process with 1, the rate of product formation
is about three orders of magnitude lower than for
AcGal. Rates of formation of transglycosylation prod-
ucts in the condensation reactions with AcGal and 1 as
a function of pH were determined by HPLC. Fig. 7
shows that the pH-optima for both substrates are
rather close.
Structures of transglycosylation products were deter-
mined after their HPLC purification from the reaction
mixture. Based on the data of 1H and 13C NMR
analysis, melting point, and optical rotation,34 the ma-
jor and minor products of transglycosylation reactions
Enzyme and enzymatic assays.—b-D-Galactosidase
from Penicillium sp. was purified according to the pro-
cedure reported in Ref. 17. Purified enzyme contained
no more than 0.05% of admixtured exoglucosidase ac-
tivity, measured with corresponding a/b-PNP gly-
cosides.38 One unit of b-galactosidase activity towards 2
was defined as the amount of the enzyme required to
hydrolyze 1 mmol of the substrate per 1 min at 37 °C in
30 mM sodium acetate buffer, pH 5.0. The activity of
b-galactosidase in the hydrolysis of 1 was determined
by measuring the concentration of the reducing sugar
during the reaction by Somogyi–Nelson’s visualization
method39 and, in the case of the hydrolysis of lactose,
by the glucose oxidase method.40 Determination of the
with 1 were identified as methyl b-
(1“6)-b- -galactopyranoside and methyl b-
actopyranosyl-(1“3)-b- -galactopyranoside, respec-
D
-galactopyranosyl-
D
D
-gal-
D
tively. They were obtained in 36% (major) and 9%
(minor) yields, respectively. These data coincide with
those for methyl b-
galactopyranoside as synthesized by transglycosylation
with b-
-galactosidase from E. coli.34 The product of
transglycosylation reactions with AcGal was b-
galactopyranosyl-(1“6)-1-O-acetyl-b- -galactopyran-
D
-galactopyranosyl-(1“6)-b-
D-
D
b-D-galactosidase activity in the hydrolysis of AcGal
D
-
was carried out after the reaction has been terminated
by freeze-drying the sample. Then the mixture was
analyzed by HPLC on a Lichrosorb-NH2 column
(Pharmacia) with refractometric detection, using iso-
cratic elution with 4:1 MeCN–water; a quantitative
interpretation was accomplished by integrating the
D
ose. Minor products of the substrate transglycosylation
were in trace amount. Acid hydrolysis of the main
product gave b-
D
-galactopyranosyl-(1“6)-D-galacto-
pyranose which was identified by TLC, HPLC, and 13C
NMR.36 The b(1“6) configuration of the galactosyl
peaks for
The effect of pH on the b-
D
-galactose and AcGal.
-galactosidase activity
bonds has been reported for only a few b-
D
-galactosi-
D
dases.36 The products of enzymatic syntheses with other
was measured at 37 °C over the pH range 3–9 in 100
mM sodium citrate buffer. The values for KM and kcat
were determined based on the initial rates of the hy-
drolysis of 1, 2, AcGal, and lactose. The hydrolysis was
carried out in 30 mM NaOAc buffer, pH 5.0, at 37 °C
and concentrations of 1 from 20 to 2 mM; 2 ranging
from 2 to 0.2 mM; AcGal from 10 to 1 mM, and
b- -galactosidases were b(1“3)- and b(1“4)-
D
galactooligosaccharides.9–11,34
Further investigations will be focused on AcGal as a
potentially effective donor in the substrate transglyco-
sylation reactions for production of different biologi-
cally active galactooligosaccharides.

640
A.I. Zinin et al. / Carbohydrate Research 337 (2002) 635–642
lactose from 20 to 5 mM. Data were analyzed by the
Lineweaver–Burk method. The inhibition constants
were measured by the Dixon method, varying the con-
centration of the substrate in the reaction mixture.41 As
an alternative, the kinetics of AcGal hydrolysis were
Hz, H-1%), 3.93 (m, 1 H, H-5%), 3.84 (dd, 1 H, J3,4 3.4,
J4,5 0.6 Hz, H-4), 3.78 (m, 3 H, CH2, H-4%), 3.66 (dd, 1
H, J5,6 7.9, J6,6 11.6 Hz, H-6%), 3.63 (dd, 1 H, J5,6 4.3
Hz, H-6%), 3.57 (ddd, 1 H, H-5), 3.52 (dd, 2 H, J2,3 9.9
Hz, H-3, H-3%), 3.45 (s, 3 H, O-Me), 3.40 (dd, 1 H,
H-2%), 3.38 (dd, 1 H, H-2).
1
studied by H NMR techniques as described next.
Acid hydrolysis of substrates.—Acid hydrolysis of 1
and AcGal was carried out at 80 °C in HCl in the
concentration range of 16.7–83.3 mM for AcGal and
100–800 mM for 1. The mixtures contained 13.5 mM
of 1 or 2.5 mM of AcGal, along with the appropriate
amount of HCl and were incubated for 16 or 4 min,
respectively. Subsequently, the reaction samples were
lyophilized and analyzed on a Lichrosorb-NH2 column
with the refractometric detection already described.
Values for k+H were calculated based on Eq. (4):
Methyl i-
D
-galactopyranosyl-(1“3)-i-D-galactopy-
ranoside.—Mp 201–202 °C, [h]D +24.5° (c 1, water);
13C NMR (D2O): l 104.32 (C-1), 103.43 (C-1%), 82.41
(C-3%), 75.03 (C-5), 74.74 (C-5%), 72.50 (C-3), 71.02
(C-2), 69.81 (C-2%), 68.55 (C-4), 68.43 (C-4%), 60.92 (C-6,
1
C-6%), 57.09 (O-Me); H NMR (D2O): l 4.47 (d, 1 H,
J1,2 7.6 Hz, H-1), 4.24 (d, 1 H, J1,2 8.0 Hz, H-1%), 4.07
(dd, 1 H, J4,3 3.4, J4,5 1.1 Hz, H-4%), 3.79 (dd, 1 H, J3,4
3.3, J4,5 1.0 Hz, H-4), 3.68 (dd, 1 H, J2,3 9.8 Hz, H-3%),
3.67 (dd, 1 H, J5,6 7.9, J6,6 11.7 Hz, H-6a), 3.64 (67 (dd,
1 H, J5,6 7.7, J6,6 11.7 Hz, H-6b), 3.62 (dd, 1 H, J5,6 4.4,
J6,6 10.3 Hz, H-6%), 3.617 (dd, 1 H, J5,6 4.0, J6,6 10.3 Hz,
H-6%), 3.59 (ddd, 1 H, H-5%), 3.56 (ddd, 1 H, H-5), 3.54
(dd, 1 H, H-2%), 3.53 (dd, 1 H, J2,3 9.9 Hz, H-3), 3.48
(dd, 1 H, J2,3 9.9 Hz, H-2), 3.45 (s, 3 H, O-Me).
k+H =[ln([S0]/[S])]/[[HCl]~]
(4)
where k+H is an acid hydrolysis constant; [S] is a sub-
strate concentration; [S0] is the initial substrate concen-
tration; [HCl] is the concentration of HCl, M; ~ is the
reaction time (min). Hydrolysis of b-
D-galactopyran-
osyl-(1“6)-1-O-acetyl-b- -galactopyranose was car-
i-
D
-Galactopyranosyl-(1“6)-1-O-acetyl-i-D-gal-
D
actopyranose.—This product was obtained in the same
manner.
ried out in 20 mM HCl for 12 h at 60 °C.
Transglycosylating acti6ity of the i-
D
-galactosi-
i-
D
-Galactopyranosyl-(1“6)-1-O-acetyl-i-D-gal-
dase.—Experiments on the transglycosylating activity
of b- -galactosidase were carried out in 30 mM NaOAc
actopyranose.—Mp 190–205 °C, [h]2D1 −4.3° (c 0.2,
water); 13C NMR (D2O): l 104.52 (C-1), 95.81 (C-1%),
76.64 (C-5%), 76.24 (C-5), 74.07 (C-3), 73.66 (C-3%), 72.19
(C-4%), 70.90 (C-4), 70.22 (C-6%), 70.06 (C-2%), 69.90
D
buffer, pH 5.0, or in 20 mM sodium phosphate, pH 6.0
at 37 °C. The reaction was terminated by freezing and
lyophilization. TLC was used for qualitative analysis of
transglycosylation products. A quantitative analysis of
the transglycosylation products in the case of the hy-
drolysis of 1 and AcGal was carried out by HPLC
chromatography on a Lichrosorb-NH2 column or by
1H NMR as described later. The effect of pH on the
1
(C-2), 62.46 (C-6), 21.87 (CH3-Ac); H NMR (D2O): l
5.53 (d, 1 H, J1,2 7.9 Hz, H-1%), 4.42 (d, 1 H, J1,2 7.5 Hz,
H-1).
1H NMR measurements.—To determine AcOH, Ac-
Gal, and transglycosylation product concentrations,
signals having chemical shifts at l 1.99, 5.50, and 4.43
ppm, respectively, were used. The initial hydrolysis rate
of AcGal was measured in 30 mM NaOAc buffer made
up in D2O, pD 5.0, 37 °C. Concentrations of AcGal
were in a range between 10 and 1 mM. The direct
NMR analysis of transglycosylation activities toward
AcGal and 1 was made in 30 mM sodium phosphate
buffer prepared in D2O, pD 6.0, 37 °C. Data were
acquired using 64 scans per series, including the first
four dummy scans; total acquisition time was 1 min.
b-D-galactosidase transglycosylation activity was mea-
sured at 37 °C over the pH range 3–9 in 100 mM
sodium citrate buffer.
Methyl i-
D
-galactopyranosyl-(1“6)-i-D-galactopy-
ranoside and methyl i-
D
-galactopyranosyl-(1“3)-i-D-
galactopyranoside.—To 1 (50 mg) in 30 mM sodium
phosphate buffer (pH 6.0, 0.4 mL), 27 units of the
b-D-galactosidase were added to initiate the reaction,
which was conducted at 37 °C for 90 min. Following
termination of the reaction by freezing and lyophiliza-
tion, the transglycosylation products were isolated by
gel-permeation chromatography using a P2 column
(5×100 cm) in water followed by HPLC purification
on a Lichrosorb-NH2 column.
Stereospecificity of the AcGal hydrolysis with b-D-
galactosidase was studied as follows: the reaction was
carried out at 37 °C in 50 mM sodium phosphate buffer
in D2O, pD 6.0. The concentration of
determined from the sum of the anomeric proton sig-
nals at l 5.26 and 4.57 ppm (a and b anomer, respec-
tively). The reaction was monitored by collecting H
NMR spectra once per min after the enzyme addition.
Anomeric proton signals (a and b) were registered
during the course of the reaction.
D-galactose was
Methyl i-
D
-galactopyranosyl-(1“6)-i-D-galactopy-
ranoside.—Mp 210–212 °C, [h]2D1 −9.5° (c 0.2, water);
13C NMR (D2O): l 104.33 (C-1), 103.81 (C-1%), 75.67
(C-5), 74.32 (C-5%), 73.22 (C-3), 73.15 (C-3%), 71.26
(C-2), 71.15 (C-2%), 69.48 (C-6%), 69.21 (C-4%), 69.14
1
1
(C-4), 61.50 (C-6), 57.85 (O-Me); H NMR (D2O): l
4.33 (d, 1 H, J1,2 7.7 Hz, H-1), 4.21 (d, 1 H, J1,2 7.71

A.I. Zinin et al. / Carbohydrate Research 337 (2002) 635–642
641
1-O-Acetyl-2,3,4,6-tetra-O-benzyl-i-
D
-galactopyran-
Research and the grant of Sixth Expertise-Competition
from the Presidium of Russian Academy of Sciences.
ose.—Glacial AcOH (0.25 mL, 4.37 mmol) was added
during 3 min to a stirred solution of 2,3,4,6-tetra-O-ben-
zyl-a-D
-galactopyranosyl 2,2,2-trichloroacetimid-ate23
(944 mg, 1.38 mmol) in anhyd CH2Cl2 (10 mL) under
argon at −20 °C. The reaction mixture was allowed to
warm to rt (25 °C) and was kept at rt for 2 h to complete
the reaction (TLC). The resulting solution was diluted
with CHCl3 and washed with satd aq NaHCO3. The
organic layer was dried by passing through a pad of
Na2SO4–Celite and concentrated at reduced pressure.
Column chromatography (0“10% EtOAc in PhH) af-
forded 610 mg (76%) of a syrup containing the title
compound, along with its a anomer (10:1 b/a by NMR).
Crystallization from Et2O–hexanes yielded 410 mg
(51%) of pure title compound: mp 102–103 °C; [h]D28
+3.8° (c 2.0, CHCl3); Rf 0.38, 9:1 (v/v) PhH–EtOAc;
1H NMR (CDCl3): l 7.45–7.25 (m, 20 H, 4×C6H5),
5.60 (d, 1 H, J1,2 8.0 Hz, H-1), 4.96, 4.86, 4.76, 4.74, 4.65,
4.46, 4.41 (8 H, 8×CH2-Bn), 4.00 (dd, 1 H, J4,5 1.2 Hz,
H-4), 3.98 (dd, 1 H, J2,3 9.7 Hz, H-2), 3.72 (m, 1 H, J5,6a
7.6, J5,6b 5.5 Hz, H-5), 3.64 (dd, 1 H, J6a,6b 8.9 Hz, H-6a),
3.63 (dd, 1 H, J3,4 2.7 Hz, H-3), 3.59 (dd, 1 H, H-6b),
2.04 (s, 3 H, CH3-Ac). 13C NMR (CDCl3): l 94.31 (C-1),
82.42 (C-3), 78.19 (C-2), 74.09 (C-4), 73.13 (C-5), 67.96
(C-6), 21.00 (CH3-Ac), 75.30, 74.70, 73.51, 72.89 (4×
CH2-Bn). Anal. Calcd for C36H38O7: C, 74.21; H, 6.57.
Found: C, 73.96; H, 6.65.
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1-O-Acetyl-i-
1-O-acetyl-2,3,4,6-tetra-O-benzyl-b-
D
-galactopyranose.—A suspension of
-galactopyranose
D
(360 mg, 0.618 mmol) in 95% EtOH (10mL) was hydro-
genated under atmospheric pressure at 36 °C over 10%
Pd–C (125 mg) for 12 h. The catalyst was removed by
centrifugation and washed with 95% EtOH. The com-
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EtOAc to give the title compound (136 mg, quant.): mp
186–191 °C (with partial decomposition), [h]3D0 +15.6 (c
1.5, MeOH), Rf 0.38, 4:1 (v/v) EtOAc–MeOH; 1H
NMR (D2O): l 5.53 (d, 1 H, J1,2 8.0 Hz, H-1), 4.01 (dd,
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Acknowledgements
The present work was supported in part by a grant no.
00-04-48878 from the Russian Foundation for Basic

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